Form and function are intricately linked in the development of the heart. The endocardial cushions of the atrioventricular (AV) canal and outflow tract must form into proper valves and septa in order to establish unidirectional blood flow. This requires induction and control of endocardial to mesenchymal transformation (EMT), a process that is vital to chamber septation and valve formation. Miscues in EMT-related signaling can lead to abnormal valve development and congenital heart defects. In this regard, regulation of the molecular pathways mediating differentiation and morphogenesis of mammalian cardiac valves is not well defined. The long-term goal of our research is to define mechanisms that are involved in the development of cardiac cushions and their eventual formation into valves. In this proposal, we will examine the MAPKKK, MEKK4, as a master regulator of this process. MEKK4 is a downstream effector involved with the receptor-Ras-MAPK pathway which we and others have strongly implicated in heart development. EGFR mutant mice have been shown to develop hyperplastic valves and a mutation making Shp-2 constitutively active increases the severity of this phenotype. HB-EGF stimulation of EGFR plays a negative role in regulating this pathway because HB-EGF-deficient embryos develop a hyperplastic valve phenotype similar to the EGFR mutant mice. We have shown that MEKK4 is expressed in the endocardial cushions during heart development and providing a dominant negative kinase inactive MEKK4 to an ex vivo assay for EMT significantly reduces mesenchyme outgrowth. Furthermore, mice expressing kinase inactive MEKK4 exhibit undefined heart defects. Since MEKK4 interacts with Shp-2 and ERK, key components in the propagation of EGF-ErbB signals, we will investigate the ability of MEKK4 to control activation of the ERK pathway during EMT through effects on Shp-2 and regulation by HB-EGF and EGFR. This is the first time MEKK4 has been implicated to the ERK pathway and the first proposed role for MEKK4 during heart development. Through our studies, we expect to i) determine the contribution of MEKK4 kinase activity and ii) elucidate the regulatory contribution of MEKK4 to heart valve development. This information will provide a more comprehensive understanding of normal and abnormal heart development that will be important in designing new repair strategies for congenital heart defects. [unreadable] [unreadable] [unreadable]